The present invention relates to a method of native-state histoculturing skin and to assays utilizing such method.
All commercial products that contact the skin (i.e., cosmetics, household products and pharmaceuticals) must undergo extensive testing to ensure that such products are not toxic to humans. Currently available means for evaluating the toxicity of such products are expensive, time-consuming and often involve exposing large numbers of live animals to toxic substances. The use of live animals for such toxicity testing has come under increasing criticism and scrutiny by the public, the scientific community and the government. There is a pressing need to develop skin toxicity assays which obviate the exposure of live animals to toxic substances.
Although a number of attempts to develop in vitro assays for skin toxicity have recently been reported, such systems have proven inadequate for accurately and reliably predicting the toxic effects of substances on natural skin. Many of these in vitro assays involve culturing individual cells, which have been isolated from the skin of subject animals. See, e.g., Van Brunt J. Biotechnology, 9: 136-137 (1991) and Naughton et al., Alternative Methods In Toxicology, ed. by A. M. Goldberg, 7: 183-189 (1989). Such assays based on cell culture are over simplified and inadequate, especially where putative toxins exert their effect by altering cell to cell interactions.
The model of Naughton et al. involves isolating skin fibroblasts from dermal tissue, growing such isolated fibroblasts in an appropriate medium, plating the fibroblasts onto a nylon mesh, isolating malanocytes from another skin sample, culturing such isolated malanocytes, plating the cultured malanocytes onto a fibroblast-coated nylon mesh, isolating keratinocytes and plating the isolated keratinocytes onto the malanocyte-fibroblast-nylon mesh. Not only does such a system lack an equivalence to natural skin, the use of such a technique involves the time-consuming steps of isolating of at least three different cell types, culturing each cell type individually and then co-culturing the individual cell types together on a nylon mesh. According to the authors, the minimum time required for establishment of this system ranges from about 8 to about 14 days.
In a similar fashion, other in vitro systems currently available or under development utilize individually cultured cells rather than intact skin. In some cases, cells from different organisms are combined into a single skin equivalent. Van Brunt, supra.
One laboratory has tried to establish a culturing system for intact skin, but failed to maintain skin viability for more than 24 hours. Kao at al, Toxicology and Applied Pharmacology, 81:502-516 (1985).
Despite the expenditure of time and energy by the academic and industrial scientific communities, there is no currently available skin histoculturing system, which allows cultured skin to remain viable and grow while maintaining native-state, three-dimensional architecture for prolonged times.
The present invention provides such a long-term, native-state histoculturing system for skin and, thus, provides a solution to the pressing and long-felt need for such a system.
The present invention provides a method of native-state histoculturing a skin sample having internal and external surfaces comprising placing the skin sample on an extracellular support matrix immersed in a medium whereby the internal surface is adjacent to the matrix and the external surface is exposed above the surface of the medium and maintaining the matrix with the skin thereon under skin culturing conditions.
The extracellular support matrix is preferably a collagen-containing gel, a homopolysaccharide sponge or a combination matrix comprising a top layer of a collagen-containing gel and a bottom layer of a homopolysaccharide sponge. The collagen-containing gel is preferably gelatinized pork skin and the homopolysaccharide is preferably cellulose.
The sample of skin utilized in accordance with the present invention is preferably of mammalian origin and, more preferably of human origin.
The present invention further provides an extracellular support matrix for native-state histoculturing skin comprising a top layer of a collagen-containing gel and a bottom layer of a homopolysaccharide sponge.
Still further, the present invention provides a skin toxicity assay comprising the steps of (a) culturing a skin sample having internal and external surfaces on an extracellular support matrix immersed in a medium under skin culturing conditions whereby the internal surface is adjacent to the matrix and the external surface is exposed above the surface of the medium, (b) contacting the skin sample with a putative toxin, (c) maintaining the skin sample and matrix in the presence of the toxin for a preselected period of time, and (d) assessing the viability of the skin sample and thereby the skin toxicity of the toxin.
Viability is assessed by measuring the incorporation into cells of the skin sample of an indicator specific for viable or dead cells. The indicators are preferably dyes capable of optical detection and, more preferably fluorescent dyes. Alternatively, viability is assessed by adding a first dye specific for cell viability and a second dye specific for dead cells to the medium, maintaining the dye-containing medium for a preselected period of time, and optically scanning the skin sample to quantify distribution of the first and second dyes in the skin sample and thereby assessing toxicity.
In a preferred embodiment, the skin toxicity assay further comprises assessing the viability of the skin sample prior to contacting the skin sample with a putative toxin and comparing the assessed viability before and after contact with the putative toxin and thereby the toxicity of the toxin.
The present invention also provides a hair growth assay comprising (a) culturing a skin sample capable of growing hair and having internal and external surfaces on an extracellular support matrix immersed in a medium whereby the internal surface is adjacent to the matrix and the external surface is exposed above the surface of the medium, (b) making a first determination of hair growth status, (c) maintaining the skin sample and matrix in the medium for a preselected period of time, (d) making a second determination of hair growth status, and (e) comparing the first and second determinations of hair growth status thereby determining hair growth.
Hair growth status is preferably a physical dimension of the hair such as hair length.
One advantage of the present invention is the provision of a histoculturing system for skin whereby the native-state, three-dimensional architecture of skin is maintained during culture.
Another advantage of the present invention is the provision of a simple, inexpensive and reliable skin toxicity assay for determining the effects of putative toxins on skin. Such an assay also provides an economically efficient means for evaluating in vitro, the affects of putative toxins, which assay does not involve exposing live animals to the toxins. The assay provides a model for studying cell viability and biochemical response in dermatological research and testing.
Yet another advantage of the present invention it the provision of a simple, inexpensive and reliable means for assessing hair growth in vitro and for measuring effects of compositions on hair growth.